Isoflavones are a group of oestrogenic compounds which belong to the flavonoid class of plant secondary metabolites. These compounds are produced naturally in certain plants expressing the enzyme isoflavone synthase and in particular in leguminous plants. The presence of isoflavones is known to provide several advantages including the facilitation of antimicrobial plant defences and establishing bacterial or fungal symbioses within plants as well aiding nitrogen fixation in root nodules.
In addition to the advantages that are conferred to plants, the dietary presence of isoflavones is also believed to provide benefits to human health. For example, dietary isoflavones are believed to be effective at reducing the risk of cancer and cardiovascular disease.
At present, in the human diet the only sources of isoflavones are certain legumes, such as soybean or chickpea. Soy constitutes by far the major dietary source, however supplementation of food products with soy or soy-extracts may adversely affect the flavour profile. It would therefore be desirable to extend the range of plants or plant tissues capable of providing an effective source of isoflavones to the human diet and in particular a source which does not adversely affect the flavour profile of a product.
WO 00/53771 teaches that to form the isoflavone daidzein in transgenic plants that do not possess an isoflavonoid pathway and thus do not produce isoflavones in nature, it would be necessary to introduce therein three new genes, namely chalcone reductase (CHR) to co-act with chalcone synthase (CHS) to form 2′,4′,4′-trihydroxychalcone, a suitable chalcone isomerase (CHI) to convert 2′,4′,4′-trihydroxychalcone to liquiritigenin, and isoflavone synthase (IFS).
The applicants have now found that the approach disclosed in WO 00/53771 does not allow the formation of daidzein in respect of many plants. Furthermore the applicants believe that the transformation of the tomato plant as exemplified in example 3 of WO 00/53771 is most unlikely to produce tomatoes with increased levels of daidzein as purported to be achieved therein.
Studies carried out by Yu et al., (Production of isoflavones genistein and daidzein in non-legume dicot and monocot tissues. Plant Physiol. 2000 124:781-793) applied transcription factors C1 and R with the co-introduction of CHR and IFS into non-differentiated Black Mexican Sweet (BMS) maize cultures. This approach yielded only trace elements of daidzein in a single cell line.
The use of this single cell system in drawing any conclusions relating to enzymology and regulation of secondary metabolic pathways in differentiated tissues is recognised in the art as unreliable (Stafford H. A. (1990) CRC Press, Boca Raton, Fla. p. 225-239).
BMS maize cell cultures are undifferentiated and are not active in flavonol biosynthesis.
The objective technical problem to be solved by the present invention therefore relates to the need to provide novel plants which comprise significantly increased levels of daidzein and/or daidzein derivatives.
It has now been found that the solution to this problem lies in a process which selects a non-isoflavone producing plant or part thereof comprising both active anthocyanin and flavonol pathways and alters said plant to increase the enzyme activity of chalcone reductase and isoflavone synthase therein.
At the time of filing it was not known that the selection of a non-isoflavone producing plant comprising both active anthocyanin and flavonol biosynthetic pathways in combination with an increase in these specific enzyme activities could be used to provide plants with increased levels of daidzein and/or derivatives thereof.
Definition of Terms
A non-isoflavone producing plant is suitably defined by the absense of isoflavone synthase enzyme activity which renders the tissues of the plant unable to produce isoflavones. The absence of isoflavone synthase activity can be determined by achieving a negative result in a standard enzyme assay as disclosed in Jung et al., (Nature Biotech. Vol. 18 Feb. 2000 p. 208-212) incorporated herein by reference.
The term ‘plant or part thereof’ is used herein to refer to an entire plant or differentiated group of cells forming a part thereof. A part of a plant for the purpose of the invention may relate to leaves, stems, fruit, seeds, flowers, roots, tubers.
The expression ‘increasing’ is used in comparison to an equivalent unmodified plant or part thereof and may be on an absolute dry weight basis or in relative terms. Except for the modifications introduced by the process of the invention, this equivalent plant is genetically identical thereto.
Daidzein as used herein is taken to comprise 7,4′-dihydroxyisoflavone. Derivatives of daidzein are taken to comprise those molecules which result from the cellular biochemical modification of daidzein. Preferably a daidzein derivative is selected from the group comprising pterocarpans e.g. medicarpin, glyceollin, isoflavanones e.g. vestitone, rotenoids e.g. munduserone, isoflavans e.g. vestitol, α-methyldeoxybenzoins e.g. angolensin, 2-arylbenzofurans e.g. centrolobofuran, isoflavonols e.g. 7,2′-dihydroxy-4′-methoxyisoflavonol, isoflav-3-enes e.g. haginin, 3-arylcoumarins e.g. glysyrin, coumestans e.g. coumestrol, coumaronochromones e.g. lupinalbin, coumaranochromene e.g. pachyrrhisomene.
Derivatives of daidzein may also result from one or more chemical processes selected from the group comprising methylation, glycosylation, prenylation and ether linkage.
A plant or part thereof that is active in anthocyanin biosynthesis has an active anthocyanin pathway and is taken to comprise a tissue which comprises mRNA encoding one or more enzymes selected from the group comprising dihydroflavonol reductase, proanthocyanidin synthase, and UDP-glucose:flavonoid-3-O-glucosyltransferase.
For the purpose of the present invention active anthocyanin biosynthesis may be determined in a plant tissue by a spectrophotometric test wherein absorbance of a hydrolysed plant extract at λvis-max 480-560 nm is indicative of an active anthocyanin pathway. Plant tissues are ground in liquid nitrogen and extracted with 80% (v/v) ethanol at 100 mg/700 μl for 30 min at room temperature (˜22/C). Following extraction the cell debris is removed by filtration through a 0.45 μm Millex_HV filter unit (Millipore Corp, USA). The ethanol extract (360 μl) is mixed with 12M HCl (40 μl). The acidified ethanol extract is assayed by spectrophotometer and absorbance determined as Aλvis-max 480-560nm, with the A657 subtracted.
It is preferred that a plant or the part thereof that is active in anthocyanin biosynthesis contains more than 10 mg/kg fresh weight anthocyanin, more preferably at least 100 mg/kg, further preferred at least 1000 mg/kg and most preferred from 1000 to 10,000 mg/kg fresh weight. Suitably this is calculated from absorption values according the formula C=A*MW*103*DF/(ε*1) in which C refers to concentration, A refers to absorption (as defined above); MW is molecular weight; DF is dilution factor; ε is molar extinction coefficient (29,600 for cyanidin 3-glucoside the major anthocyanin in nature) and l is the path length.
A plant or part thereof which is active in flavonol biosynthesis has an active flavonol pathway and is taken to comprise any tissue which comprises mRNA encoding one or more enzymes selected from the group comprising chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonol synthase.
For the purpose of the present invention whether a plant is active in flavonol biosynthesis may be determined by preparing a hydrolysed tissue extract and detection by HPLC analysis.
For extraction, tissues are harvested and flash frozen in liquid nitrogen before being stored at −80° C. The tissues are then ground to a fine powder to ensure a homogeneous mix. An aliquot from this mixture is then extracted for 30 min at room temperature (˜22° C.) in 80% (v/v) ethanol at 100 mg/700 μl. Following extraction, the cell debris is removed by filtration through a 0.45 μm Millex-HV filter unit (Millipore Corp, USA). The filtrate is stored at −20° C. prior to HPLC analyses.
For hydrolysed extracts, 40 μl of 12M HCl is added to 360 μl from each tissue extract, before incubating at 90° C. for 40 min.
After hydrolysis, an aliquot from each extract is filtered through a 0.2 μm PTFE disposable filter (Whatman). The filtrate (20 μl) is injected into the HPLC system (HP1100, Agilent) via an autosampler maintained at 4° C. The analytical column (Prodigy Phenyl-3, 4.6×150 mm, particle size 5 μm, (Phenomenex) is held at 30° C. Detection is by diode array, monitoring at 262, 280, and 370 nm. Observed peaks are scanned from 210-550 nm to obtain spectra. Chemstation software (Rev. A.8.03) was used to control the system and collect and analyse data.
Absorbance spectra (corrected for baseline spectrum) and retention time of peaks are compared with those of commercially available flavonol standards to determine whether the plant tissue is active in flavonol biosynthesis.
It is preferred that a plant or the part thereof that is active in flavonol biosynthesis contains at least 10 mg/kg fresh weight of flavonol, preferably at least 100 mg/kg more preferred at least 1000 mg/kg, most preferred from 1000 to 10000 mg/kg.
A ‘functional equivalent’ nucleotide sequence is any sequence which encodes a protein which performs the same biological function.
According to another embodiment, a functionally equivalent nucleotide sequence shows at least 50% identity to the respective DNA sequence. More preferably a functionally equivalent DNA sequence shows at least 60%, more preferred at least 75%, even more preferred at least 80%, even more preferred at least 90%, most preferred 95-100% identity, to the respective DNA sequence (DNAStar MegAlign Software Version 4.05 and the Clustal algorithm set to default parameters).
According to a further preferred embodiment a functionally equivalent sequence shows not more than 5 base pairs difference to the respective DNA sequence, more preferred less than 3, e.g. only 1 or 2 base pairs different.
According to another embodiment a functionally equivalent sequence is capable of hybridising under low stringent (2×SSC, 0.1% SDS at 25° C. for 20 min) conditions to the respective sequence, more preferably a functionally equivalent sequence is capable of hybridising under medium stringent conditions (1×SSC, 0.1% SDS, 25° C. for 20 min), further preferred a functionally equivalent sequence is capable of hybridising under high stringent conditions (0.1×SSC, 0.1% SDS, 25° C. for 20 min).
Preferably an equivalent DNA sequence is capable of transcription and subsequent translation to an amino acid sequence showing at least 50% identity to the amino acid sequence encoded by the respective DNA sequence. More preferred, the amino acid sequence translated from an equivalent DNA sequence has at least 60%, more preferred at least 75%, even more preferred at least 80%, even more preferred at least 90%, most preferred 95-100% identity to the amino acid sequence encoded by the respective DNA sequence (DNAStar MegAlign Software Version 4.05 and the Clustal algorithm set to default parameters.)